1. Field of the Invention
The present invention relates generally to a method for measuring the ratio of analytes in a sample. The methods are generally applicable to any pair of analytes in which the result is expressed as a ratio. Additionally, multiple ratios can be expressed as ratios of multiple pairs of analytes
2. Description of the Prior Art
Computing the ratio of two or more analytes in a sample is a useful comparison in a variety of contexts. Ratios are frequently used to compensate for variability in the composition or concentration of the analytes in a sample. For example, when measuring analytes present in urine, the concentration of analytes in any given sample can vary significantly. These concentration fluctuations can be normalized by creating a ratio between different analytes present in a sample. For example, a ratio can be created between the concentration of a urinary analyte and the concentration of creatinine in urine. Creating this relationship between two analytes in the sample allows for a more meaningful measurement in light of the concentration fluctuations. It is also possible to normalize the concentration of a desired analyte in tissue against the concentration of total protein or a specific protein such as, for example, albumin. Ratios can also compensate for correlated errors in the measurement, e.g., dilution errors.
A brief description of the most common prior method for calculating the concentration ratio of analytes is illustrated in FIG. 1.
At step 101 a specimen is taken from a patient. Example specimens include blood, urine, or other bodily fluids. The specimen forms the basis for the sample from which a ratio of analytes will be measured and computed. The specimen does not necessarily have to come directly from a patient. The specimen may be available from another source.
At step 102, the specimen is converted into a pre-processed sample. A pre-processed sample can also be referred to as a test sample. This step may consist of anything done to the specimen before any analyte measurements are taken. For example, in certain embodiments, the specimen may be sampled and diluted by a specific amount. In other embodiments, an anti-coagulant may be added to the specimen. This step is optional as there may be times when the entire unaltered specimen will be used as the pre-processed sample.
The sample is measured in steps 103 and 104. In these two steps, the quantities of interest in the sample are presented as responses. Step 103 consists of all the processing done by the instrument to ready the pre-processed sample for reading. Processing the sample can involve a variety of steps. Using a multi-analyte sandwich immunoassay as an example, the sample is reacted with antibodies or specific binding reagents attached to a solid phase, the solid phase is washed to remove unbound material, reacted with one or more labeled antibodies or binding reagents, and washed to remove unbound labeled reagent for subsequent reading of the response. Other examples include high-performance liquid chromatography (HPLC), immunoassay, electrophoresis, capillary electrophoresis, ultra-violet/visible/infrared spectroscopy, raman spectroscopy, surface enhanced raman spectroscopy, mass spectroscopy, gas chromatography, or others. There may be instances when no processing steps need to be taken on the sample.
In Step 104, the selected reading technique is used to determine the response of each analyte in the sample that is to be used in the ratio. Step 104 is referred to as reading the analyte (responses). Reading techniques that may be appropriate include: fluorescence, absorbance, reflectance, ion current, electrochemical potential, optical density, color, surface plasmon resonance, or others. The processing and reading techniques chosen usually depends on the nature of the sample and analytes to be measured. The response of the analytes measured using the chosen measurement method can be in any unit that is appropriate for the selected method. These units could be fluorescence units, optical density, color, ion current, chemiluminescence units, electrical signal, or others. The analytes can also be measured in a multiplexed format, where many analytes are measured in a single pass, or they can measured in multiple, but separate individual assays.
The analyte responses are then converted into their corresponding concentrations. This step is shown at steps 105 and 106. Typically this conversion is accomplished using a device such as a calibration curve.
Each analyte response must be individually converted into a concentration value before a ratio of the analyte concentrations is calculated. A concentration value for an analyte can depend not only on the measured response of the given analyte, but also on the responses of other analytes in the sample because of cross-interactions such as cross-reactivity between analytes. The relationship between the response of an analyte in a sample measured using the selected measuring technique and the analyte's concentration in the same sample is typically nonlinear. Because of this non-linear relationship, the ratio of the analyte responses can be quite different than the ratio of the analyte concentrations. Therefore, it is necessary to compute the concentration of each analyte individually before computing the ratio if the ratio of the concentration of the analytes is to be computed.
A calibration curve is typically used to convert an analyte's response into a concentration value. A calibration curve for an analyte can be created by choosing a model for the curve and then determining the values of the model coefficients by measuring analyte responses from a set of samples where the concentration of the analyte is known before any measurement is taken. The analyte responses obtained from these known samples may be graphed to produce a calibration curve that is then used to infer what the concentration of an analyte is in a sample where the concentration is unknown. A separate calibration curve has to be created for each analyte response that is to be converted into a concentration value. In practice, the equation for the model with determined coefficient values may be used directly to determine the concentrations without graphing. Reference to a calibration curve is generally used because it is more illustrative than reference to a calibration equation.
At step 107 the individual concentrations calculated using the calibration curves are used to determine the desired analyte ratio. This can be done through a simple mathematical operation such as division. Ratios of analytes are typically unitless.
It is also sometimes necessary to adjust the ratio obtained in step 107 in order to make the measured ratio match what the ratio would have been using a different measuring technique. This step is shown at step 108. This extra conversion is done because it is often customary for a ratio to be presented as if the ratio was determined using a specific measuring technique, and this specific technique may not be the same technique used in steps 103 and 104. There may be well-known differences between the ratio of analytes obtained through these different measurement techniques, and the values can be converted between the two formats. Step 108 is an optional step depending on how the specific analyte ratio is to be ultimately presented. Additional information on standardized ratio measurements can be found in “Proposed changes for reporting HbA1c”, IVD Technology (May 2007) and “Implementation of Standardization of Hemoglobin A1c Measurement”, Clinical Chemistry, 54:6, 1098-1099 (2008). Both references are hereby incorporated in their entirety for all purposes.
This known method for determining the ratio of analytes has a number of problems. Interactions between the analytes in the sample can introduce inaccuracies in the known method. The response of one analyte can influence and change the response of the second analyte. For instance, in some measurement techniques, such as immunoassay, the first antibody used to measure the first analyte response may also interact with the second analyte in the sample. This cross-reaction would require a different calibration curve for the first analyte for each different value of the second analyte. A single average calibration curve could be created for the first analyte, but this would introduce inaccuracies in the responses obtained for that analyte. In addition, the second antibody could also react with the first analyte in the sample. This would compound the problem.
Another problem with the known method is that a calibration curve needs to be created and maintained for each analyte. This involves the choice of an appropriate calibration model and calibrators for each curve.
Another problem with the known method is that there is less opportunity to cancel noise that is correlated at the response level. The cancellation is done only during the determination of the ratio of the concentrations.
The proposed method for determining the ratio of analytes improves on the prior methods of determining the ratio of analytes by no longer directly using individual analyte concentrations as a part of the method. In addition to being more convenient to use, the proposed method produces more accurate analyte ratios because the proposed method inherently handles interferences introduced into the readings from interactions between analytes in a sample. It may offer additional advantages by requiring only a single calibration model and better cancellation of noise correlated at the response level.